Role of Brain Derived Extracellular Vesicles in Decoding Sex Differences Associated with Nicotine Self-Administration

Figures

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  Figure 1. Scheme of experimental in vivo design. A visual representation of the experimental process beginning with nicotine self-administration followed by brain isolation, EV extraction, and proteomics analysis.

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  Figure 2. Validation of BDEV purity. (A) Western blot analysis on BDEV isolated from a control saline animal shows the expression of the positive markers CD81, flotillin-1, and Hsp70. (B) The negative marker calnexin was enriched in the whole tissue lysate (L) but absent in the EVs fraction (EV). (C) Nano tracking analysis (NTA) shows an increase in BDEV particle concentration in both and male and female rats exposed to nicotine. However, no significant changes in concentrations of isolated BDEVs from the two treatment groups between the sexes was observed as determined by a 2-way ANOVA followed by Sidak’s multiple comparison test.

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  Figure 3. Characterization of BDEV. (A) BDEV isolated using a sucrose density gradient from the three groups were characterized using TEM that revealed increased sizes in both males and females which was larger in the latter post nicotine treatment. NTA analysis showing the different size distribution. (B) Average BDEV sizes. Average BDEV sizes for each group is represented as mean ± SEM, * p < 0.05 as determined by a 2-way ANOVA followed by Sidak’s multiple comparison test.

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  Figure 4. Chronic nicotine treatment increases the biogenesis and the expression of ESCRT dependent and independent pathway proteins. A custom qRT-PCR panel for EV biogenesis genes revealed several genes involved in the ESCRT dependent (complexes 0, I, II, and III) and independent pathways to be significantly upregulated in the females post nicotine self-administration. Data represented as mean ± SEM, n = 4–6 animals per group, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05 (adjusted p value) as determined by a multiple t-test followed by Holm–Sidak correction.

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  Figure 5. Differential expression of BDEV proteins in male and female rats post nicotine self-administration. (A) Venn diagram showing the differentially expressed BDEV proteins identified from the proteomics screen. (B,C) Heatmap showing the top differentially expressed proteins between the two sexes post nicotine self-administration compared to saline. MS, MN; FS, FN—males, females self-administering saline and nicotine respectively.

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  Figure 6. Mapping of biological processes. ClueGO analysis showing enriched biological processes in males and females after nicotine self-administration. The asterisks represent the group term p-value representing each category. ** p < 0.01.